Formaldehyde is frequently used to inactivate, stabilize, or immobilize proteins. The treatment results in a large variety of chemical modifications in proteins, such as the formation of methylol groups, Schiff bases, and methylene bridges. The purpose of the present study was to identify the stable formaldehyde-induced modifications in a small protein. Therefore, insulin was treated with excess formaldehyde (CH2O) or deuterated formaldehyde (CD2O). In a separate experiment, insulin was modified by formaldehyde (CH2O vs CD2O) and glycine. The mixture of CH2O-treated and CD2O-treated insulin was digested by the proteinase Glu-C. The peptide fragments obtained were analyzed by liquid chromatography-mass spectrometry (LC-MS). Seven intramolecular cross-links were identified in formaldehyde-treated insulin. Furthermore, eight out of the sixteen potentially reactive sites of the insulin molecule were modified by incubation with formaldehyde and glycine. Both the location and the chemical nature of the modifications could be assigned based on the mass increase of potential adducts as elucidated in our previous study (B. Metz et al. (2004) J. Biol. Chem. 279, 6235-6243). To confirm the assigned structures, LC-MS measurements with collision-induced dissociation (LC-MS/MS) were performed on insulin fragments. The results of the LC-MS/MS analyses agreed excellently with the assignments. The study showed that arginine, tyrosine, and lysine residues were very reactive. However, eight theoretically reactive residues did not show detectable modifications, probably because of their low intrinsic reactivity, inaccessibility, or both. The asparagine, glutamine, and histidine residues were not converted in insulin. The N-termini of insulin were partly converted to the expected imidazolidinone adducts, indicating that the protein conformation affects the accessibility and reactivity of these residues. In conclusion, this study shows that, based on our current insights in the chemistry of the reactions between proteins and formaldehyde, we are able to elucidate the location and nature of formaldehyde-induced modifications in a small protein. The approach followed in this study may be generally applicable to larger formaldehyde-treated proteins, such as toxoids used in vaccines.

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatographymass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.Aldehydes, such as formaldehyde and glutaraldehyde are widely employed reagents in the biochemical, biomedical, and pharmaceutical fields. Formaldehyde, for example, is applied to inactivate toxins and viruses for the production of vaccines, such as diphtheria, tetanus toxoid, hepatitis A, anthrax, and inactivated polio vaccine, and to stabilize recombinant pertussis toxin (1-4). The vaccine quality depends to a considerable extent upon the chemical modifications caused by the formaldehyde treatment (1, 5, 6). Formaldehyde is also used for isotope-labeling of proteins (7-9), for studying protein-protein interactions, e.g. histone organization in nucleosomes (10 -12), and for fixation of cells and tissues (13). Glutaraldehyde is utilized for the preparation of bioprostheses such as heart valves and vascular grafts (14 -16) and for conjugation of enzymes to carrier systems (17). These examples demonstrate the wide range of roles of aldehydes in the biomedical field. Besides the use of aldehydes in diverse applications, they can also destroy important sites of pro...

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.

In this article, the authors examine antecedents and consequences of the service climate in boundary-spanning self-managing teams (SMTs) that deliver financial services. Using data from members of 61 SMTs and their customers, the authors show a differential impact of the SMT service climate on various marketing performance measures. Furthermore, they obtain support for independent group-level effects of intrateam support and team member flexibility on employee perceptions of the SMT service climate. Both effects are persistent over time and demonstrate that collective perceptions in the SMT have incremental value in the explanation of the service climate.

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This empirical study of 323 new ventures examines how task and relationship conflict in the founding top management team mediates the effect of lead founder personality on new venture performance. The results reveal that (1) openness and agreeableness increase task conflict, whereas conscientiousness decreases it, and (2) openness, extraversion, and conscientiousness decrease relationship conflict, whereas neuroticism increases it. Furthermore, task conflict increases venture performance, whereas relationship conflict decreases venture performance and weakens the positive effect of task conflict. In addition, task and relationship conflict do not mediate the effect of extraversion, and they only partially mediate the effects of openness and neuroticism on new venture performance. Openness and neuroticism exert a direct impact on new venture performance, in addition to their indirect impact through task and relationship conflict.

Blood glucose, plasma insulin, and glucagon levels were measured in undisturbed and free-moving rats. The insulin and glucagon levels rise in the 1st min after the beginning of food ingestion, whereas the glucose level begins to increase only in the 3rd min if carbohydrate-rich food is eaten. This early rise in insulin and glucagon level is also observed under conditions in which carbohydrate-free food is eaten. A similar release of insulin and glucagon can be obtained by injection of 0.1 microgram of norepinephrine into the ventromedial hypothalamus, but the same injection made into the lateral hypothalamus causes release of insulin only, whereas injections in other hypothalamic areas are nearly without effect. Similar injections of isoproterenol did not cause changes in insulin, glucagon, and glucose levels. It is suggested that the early insulin and glucagon responses are of reflex origin and that the ventromedial and lateral hypothalamic areas are relay stations in the reflex pathways. The lack of effect of atropine to block the insulin and glucagon responses to noradrenergic stimulation of the ventromedial hypothalamus indicates that the efferent pathway is not cholinergic.